The role of the essential metal ions bound by Aeromonas aminopeptidase will be investigated by means of metal ion substitutions in conjunction with spectral and kinetic determinations. Mechanistic and active site studies of aminopeptidases have lagged behind those of most other classes of proteolytic enzymes, although metalloaminopeptidases and ubiquitously distributed in nature and have important biological functions. The long-term objective of the research is to formulate a reaction mechanism for this class of enzymes; the specific aims in this proposal are to delineate the roles played by each of the two zinc ions bound by Aeromonas aminopeptidase, a small, stable enzyme that is available in quantities sufficient for physical studies. Cobalt will be selectively substituted for zinc (singly and doubly) into each of the two metal binding sites and the spectra of these cobalt-aminopeptidases will be determined by absorption, circular dichroic, magnetic circular dichroic and electron paramagnetic resonance spectral measurements, to probe the ligand geometry; similar studies will be performed with copper-aminopeptidase. Studies with "hybrid ion" aminopeptidases that contain two different metal ions will be made both by kinetics and by spectral measurements. Perturbations of the spectra of cobalt- and copper-substituted aminopeptidases will be studied, as will perturbations caused by slowly hydrolyzed substrates at low temperatures. Radiationless energy transfer kinetics (pre-steady-state) of dansylated peptides will be employed to determine the minimal number of intermediates involved in catalysis by the aminopeptidase and will be used also for rapid accumulation and evaluation of steady-state kinetic data.